1. Field of the Invention
The present invention is in the field of detecting the presence of a nucleic acid sequence in a sample.
2. Description of the Background Art
Fluorescence in situ hybridization ("FISH") is widely used in medical diagnostics. Present technology for in situ hybridization includes the use of fluorescence probes such as fluorescein and rhodamine and detection of hybridized DNA by localization of the product of an enzyme catalyzed reaction.
The following references describe known fluorescence in situ hybridization techniques:
Swiger, R. R., and Tucker, J. D., "Fluorescence In Situ Hybridization: A Brief Review," Environmental and Molecular Mutagenesis 27:245-54 (1996). PA1 Schrock, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J., Ferguson-Smith, M. A., Ning, Y., Dedbetter, D. H., Bar-Am, I., Soenksen, D., Garini, Y., and Ried, T., "Multicolor Spectral Karyotyping of Human Chromosomes," Science 273:494-98 (1996). PA1 Lewis, R., "Chromosome Charting Takes a Giant Step," Photonics Spectra 48-50 (1996). PA1 Fox, J. L., Hsu, P., Legator, M., Morrison, L. E., and Seelig, S. A., "Fluorescence In Situ Hybridization: Powerful Molecular Tool for Cancer Prognosis," Clinical Chemistry 41:1554-59 (1995). PA1 Nederlof, P. M., van der Flier, S., Wiegant, J., Raap, A. K., Tanke, H. J., Ploem, J. S., and van der Ploeg, M., "Multiple Fluorescence In Situ Hybridization," Cytometry 11:126-31 (1990). PA1 Siadat-Pajouh, M., Ayscue, A. H., Periasamy, A., and Herman, B., Introduction of a Fast and Sensitive Fluorescent In Situ Hybridization Method for Single-copy Detection of Human Papillomavirus (HPV) Genome," The Journal of Histochemistry and Cytochemistry 42:1503-12 (1994). PA1 Kearns, W. G. and Pearson, P. L., "Fluorescent In Situ Hybridization Using Chromosome-Specific DNA Libraries," Methods in Molecular Biology 33:15-22 (1994). PA1 Tkachuk, D. C., Pinkel, D., Kuo, W., Weier, H., and Gray, J. W., "Clinical Applications of Fluorescence in situ Hybridization," GATA 8(2):67-74 (1991). PA1 Denijn, M., Schuurman, H., Jacobse, K. C., and Weger, R. A., "In Situ hybridization: A valuable tool in diagnostic pathology," APMIS 100:669-681 (1992). PA1 McNeil, J. A., Villnave Johnson, C., Carter, K. C., Singer, R. H., and Bentley Lawrence, J., "In Situ Hybridization," GATA 8(2):41-58 (1991). PA1 Schwarzachter, T., and Heslop-Harrison, J. S., "Direct Fluorochrome-Labeled DNA Probes for Direct Fluorescent In Situ Hybridization of Chromosomes," Methods in Molecular Biology, Vol. 28: Protocols for Nucleic Acid Analysis by Nonradioactive Proves 167-76 (1994). PA1 Durrant, J., Brunning, S., Eccleston, L., Chadwick, P., and Cunningham, M., "Fluorescein as a label for non-radioactive in situ hybridization," Histochemical Journal 27:94-99 (1995). PA1 Maestri, M., Sandrini, D., Balzani, V., Maeder, U. and von Zelewsky, "Absorption Spectra, Electrochemical Behavior, Luminescence Spectra, and Excited-State Lifetimes of Mixed-ligand Ortho-Metalated Rhodium(III) Complexes," Inorg. Chem., 26:1323-1327 (1987). PA1 Sutin, N. and Creutz, C., "Properties and Reactivities of the Luminescent Excited States of Polypyridine Complexes of Ruthenium(II) and Osmium(II)," Inorg. & Organometall. Photochem., Chap. 1, pp. 1-27 (1978). PA1 Hager, G. D., Watts, R. J. and Crosby, G. A., "Charge-transfer Excited States of Ruthenium(II) Complexes. Relationship of Level Parameters to Molecular Structure," J. Am. Chem. Soc., 97;7037-7042 (1975). PA1 Orellana, G. and Braun, A. M., "Quantum Yields of .sup.3 MLCT Excited State Formation and Triplet-Triplet Absorption Spectra of Ruthenium(II) Tris-Chelate Complexes Containing Five- and Six-Membered Heterocyclic Moieties," J. Photochem. Photobiol. A. Chem.., 48:277-289 (1989). PA1 Harrigan, R. W. and Crosby, G. A., "Symmetry Assignments of the Lowest CT Excited States of Ruthenium(II) Complexes Via a Proposed Electronic Coupling Model," J. Chem. Phys., 59(7):3468-3476 (1973). PA1 Yersin, H. and Braun, D., "Isotope-Induced Shifts of Electronic Transitions: Application to [Ru(bpy-h.sub.8).sub.3 ].sup.2+ and [Ru(bpy-d.sub.8).sub.3 ].sup.2+ in [Zn(bpy-h.sub.8).sup.3 ] (ClO.sub.4).sub.2," Chem. Phys. Letts., 179(1,2):85-94 (1991). PA1 Coe, B. J., Thompson, D. W., Culbertson, C. T., Schoonover, J. R. and Meyer, T. J., "Synthesis and Photophysical Properties of Mono(2,2',2'-Terpyridine) Complexes of Ruthenium(II)," Inorg. Chem., 34:3385-3395 (1995). PA1 Lees, A. J., "Luminescence Properties of Organometallic Complexes," Chem. Rev., 87:711-743 (1987). PA1 DeArmond, M. K. and Carlin, C. M., "Multiple State Emission and Related Phenomena in Transition Metal Complexes," Coordination Chem. Rev., 36:325-355 (1981). PA1 Kondo, T., Yanagisawa, M. and Fujihira, M., "Single Exponential Decay for the Luminescence Intensity of Ru(bpy).sub.3.sup.2+ Complex in Langmuir-Blodgett Films," Chem. Letts., 1639-1993 (1993).
None of these papers mentions the use of metal-ligand complexes in fluorescence in situ hybridization to detect the presence of a nucleic acid.
One limitation of prior art DNA hybridization is the low levels of light available from commonly used fluorophores, the presence of significant background fluorescence which limits sensitivity, and photobleaching of the probes. The resulting fluorescence is typically on a nanosecond time scale, which is also the decay time of the commonly used fluorophores. In addition, the commonly used fluorophores display small Stoke's shifts making it difficult to detect their fluorescence in the presence of a fluorescent background. There is also a need for fluorophores with greater resistance to fading and increased shelf life, i.e., archivability of the slides.
There is extensive literature regarding the spectral properties of metal-ligand complexes. The following is a list of papers regarding metal-ligand complexes:
None of the above references suggest use of metal-ligand complexes in fluorescence in situ hybridization.
There remains a need in the art for improved methods of detecting the presence of nucleic acid sequences.